This invention relates to the use of a viral enterotoxin or derivative as an adjuvant to enhance immune responses. More particularly it relates to the use of a viral enterotoxin or derivative as an adjuvant at mucosal surfaces to potentiate immune responses.
Recombinant DNA technology has stimulated the pursuit of new, safe and effective vaccines. Disadvantages of recombinant vaccines include the need for large, repeated antigen doses, and a general failure to generate major histocompatibility complex (MHC) class I-restricted immune responses. To overcome these limitations, recombinant vaccines require the use of systemic- or mucosal-active immunostimulating agents, which are referred to as adjuvants (Gradon, et al., 1999). Immunopotentiation by adjuvants can result from quantitative enhancement of or qualitative alteration of components of the immune response compared to the immune response generated by an immunogen alone. Many compounds possess adjuvant properties but the only adjuvants currently licensed for use in humans by the Food and Drug Administration are aluminum salts (aluminum hydroxide or aluminum phosphate), which are relatively weak adjuvants approved only for systemic administration.
Development of mucosal vaccines has lagged behind systemic vaccines because of our limited knowledge of mucosal immunity and because no adjuvants have been licensed for use at mucosal surfaces. Mucosal vaccines and adjuvants would be advantageous because greater than 80% of pathogens enter the host at mucosal sites. Localized infections of the mucosa are the most common cause of mortality and morbidity in humans, and many pathogens that cause systemic infections gain access to the body at mucosal sites, including HIV, measles virus and polio virus (Gradon, et al., 1999). Therefore, a vaccine strategy that can potentially prevent the initial infection of the host is likely to be more successful than one that resolves infection before the disease ensues.
The bacterial enterotoxins, cholera toxin (CT) and Escherichia coli (E. coli) heat-labile enterotoxin (LT), are the most potent mucosal adjuvants described to date, but their enterotoxicity precludes their use in humans. Enterotoxins produced by V. cholera, E. coli and Salmonella have similar modes of action. Cholera toxin is a protein consisting of three polypeptides, A1, A2, and B subunits. The B subunit contains the binding site by which the cholera toxin binds to the ganglioside (GM1), located on the cell membrane. The binding of the B subunit to the GM1 receptor facilitates the translocation of the A subunit through the membrane. The A1 subunit activates the cellular enzyme, adenylcyclase, causing conversion of ATP to cyclic AMP (cAMP). The increased levels of cAMP result in secretion of water and electrolytes into the small intestine through interactions with cAMP-sensitive NaCl transport mechanisms. It has been suggested that the adjuvanticity of CT and LT is associated with their ability to increase gut permeability; therefore, facilitating access of luminal antigens to the gut mucosal immune system (Lycke, et al., 1991). In addition to modulating the mucosal immune system by stimulating synthesis of cAMP, it has also been suggested that CT can modulate the mucosal immune response by stimulating cellular syntheses of arachidonic acid metabolites (Peterson, et al., 1999).
Recent studies have examined the potential of CT and LT as mucosal adjuvants against a variety of bacterial and viral pathogens (Xu-Amano et al., 1994; Xu-Amno et al., 1993; Yamanoto et al., 1996 and Wu, et al., 1997). However, prior art indicates that as little as 5 xcexcg of purified CT, administered orally, was sufficient to induce significant diarrhea in volunteers, while ingestion of 25 xcexcg of CT elicited a full 20-liter cholera purge (Levine, et al., 1983). Similar studies have shown that LT induces fluid secretion at doses as low as 2.5 xcexcg when administered in conjunction with a vaccine (Freytag and Clements, 1999).
A number of attempts have been made to alter the toxicity of LT and CT, most of which focus on eliminating activity of subunit A, which is associated with enterotoxicity. Recent studies have shown that site-directed mutagenesis to change any amino acid in CT or LT involved in the ADP-ribosylation results in a corresponding loss of toxicity and adjuvanticity (Yamamoto, et al., 1997 and Lycke, et al., 1992). Therefore, a logical conclusion is that ADP-ribosylation and induction of cAMP are essential for the enterotoxicity and adjuvanticity of LT and CT. As a result, a linkage has been established between enterotoxicity and adjuvanticity. Thus, there is a need for new, effective systemic and mucosal adjuvants suitable for human use to enhance the efficacy of vaccines to prevent life-threatening infections.
Although there are no sequence homologies between the rotavirus non-structural protein (NSP4) and any known bacterial enterotoxin, NSP4 by itself is immunogenic and antibodies against NSP4 protect neonatal mice against diarrhea induced by a rotavirus challenge (Ball, et al., 1996, and Zeng and Estes 1999). The mechanism of adjuvanticity of NSP4 and CT or LT mutants may be different. CT and LT activate cAMP which is required for adjuvanticity and enterotoxicity (Freytag and Clements, 1999, and Cheng, et al., 1999). In contrast, NSP4 enterotoxigenic activity results from chloride secretion stimulated by a signal transduction pathway that increases intracellular calcium through receptor-mediated phospholipase C (PLC) activation and inositol 1,4,5-triphosphate (IP3) (Ball, et al., 1996, and Dong, et al., 1997, and Morris et al., 1999). Activation of B and T lymphocytes also involves PLC and IP3 that stimulate increases in intracellular calcium (Freytag and Clements, 1999).
This invention demonstrates for the first time the use of NSP4 as a new adjuvant. It is noteworthy that although the prior art has used bacterial and aluminum compounds as adjuvants for potentiating an immune response, the use of a viral enterotoxin or derivative as an adjuvant has gone unrealized, suggesting that this invention is indeed novel and nonobvious.
An embodiment of the present invention is a method of potentiating an immune response against an antigen in an animal comprising the step of administering the antigen and an adjuvant wherein the adjuvant is a rotavirus enterotoxin or derivative thereof. Exemplary rotavirus enterotoxins include, but are not limited to the NSP4 group A genotypes A, B, C or D.
In specific embodiments, the adjuvant can be either a toxin or a non-toxic derivative. More particularly, a derivative of rotavirus can include, but is not limited to OSU NSP4-P138S or SA11 NSP4 aa 112-175.
In a preferred embodiment of the present invention, the antigen and the adjuvant are administered to an animal using standard methods. They can be co-administered or administered separately. Administration may be mucosal (e.g., intranasal, ocular, gastrointestinal, oral, rectal and genitourinary tract) or parenteral (e.g., intraperitoneal, intravenous, subcutaneous or muscular.) Animals that may be treated using the method of the invention include, but are not limited to humans, cows, horses, pigs, dogs, cats, sheep goats, rabbits, rats, mice, birds, chickens or fish.
Yet further, in specific embodiments, the immune response is systemic or mucosal.
A further embodiment of the present invention is that the antigen is rotavirus-like particles or influenza A. Additional antigens that can be used in the present invention include, but are not limited to cancer vaccines, viral vaccines, bacterial vaccines or parasitic vaccines.
A specific embodiment of the present invention is a method of potentiating an immune response against influenza A virus by administering to a mucosal surface an inactivated influenza A virus and an adjuvant. Specifically, the adjuvant is a rotavirus enterotoxin or a derivative thereof.
Another specific embodiment of the present invention is a method of potentiating an immune response to rotavirus-like particles by administering to a mucosal surface rotavirus-like particles and an adjuvant. In specific embodiments, the adjuvant is a rotavirus enterotoxin or a derivative thereof.
Other and further objects, features and advantages would be apparent and eventually more readily understood by reading the following specification and by reference to the company drawings forming a part thereof, or any examples of the present preferred embodiments of the invention are given for the purpose of the disclosure.